Antiviral Effects of Clinically-Relevant Interferon-α and Ribavirin Regimens against Dengue Virus in the Hollow Fiber Infection Model (HFIM)

Abstract

Dengue virus (DENV) is the most prevalent mosquito-borne viral illness in humans. Currently, there are no therapeutic agents available to prevent or treat DENV infections. Our objective was to fill this unmet medical need by evaluating the antiviral activity of interferon-α (IFN) and ribavirin (RBV) as a combination therapy against DENV. DENV-infected Vero and Huh-7 cells were exposed to RBV and/or IFN, and the viral burden was quantified over time by plaque assay. Drug-drug interactions for antiviral effect were determined by fitting a mathematical model to the data. We then assessed clinically-relevant exposures of IFN plus RBV using the hollow fiber infection model (HFIM) system. RBV monotherapy was only effective against DENV at toxic concentrations in Vero and Huh-7 cells. IFN, as a single agent, did inhibit DENV replication at physiological concentrations and viral suppression was substantial in Huh-7 cells (Half maximal effective concentration (EC₅₀) = 58.34 IU/mL). As a combination therapy, RBV plus IFN was additive for viral suppression in both cell lines; however, enhancement of antiviral activity at clinically-achievable concentrations was observed only in Huh-7 cells. Finally, clinical exposures of RBV plus IFN suppressed DENV replication by 99% even when treatment was initiated 24 h post-infection in the HFIM. Further evaluation revealed that the antiviral effectiveness of the combination regimen against DENV is mostly attributed to activity associated with IFN. These findings suggest that IFN is a potential therapeutic strategy for the treatment of DENV.

Keywords: Dengue virus; antiviral therapy; combination therapy; hollow fiber infection model; interferon-α; mathematical modeling; ribavirin.

Figure 1

Antiviral activity of ribavirin (RBV) and interferon-α (IFN) as a combination therapy against DENV2. (A) Vero cells were infected at a multiplicity of infection of 0.001 PFU/cell and exposed to RBV and/or IFN. Viral supernatants were collected on day 3; (B) Huh-7 cells were infected at a multiplicity of infection of 0.01 PFU/cell and exposed to RBV and/or IFN. Supernatant samples were harvested after two days post-infection. Viral burden was quantified by plaque assay in Vero cells and reported as log₁₀ PFU/mL. Data points correspond to the geometric mean of three independent samples and error bars represent one standard deviation. Horizontal dashed lines represent the assay limit of detection.

Figure 2

The influence of time to initiation of treatment on the antiviral effect of ribavirin (RBV) plus Interferon-α (IFN) when administered at exposures associated with the clinical regimens against DENV2 in the Hollow fiber infection model (HFIM) system. Huh-7 cells were mixed with DENV at a multiplicity of infection of 0.001 PFU/cell and inoculated in the HFIM. Drug exposure was the same between all treatment arms, with the exception of the control (untreated) arm. The combination regimen was administered into HF cartridges at different times post-inoculation (0 h, 2 h, 6 h, 12 h, and 24 h). Supernatant samples were collected from the system for three days following drug administration and viral burden was quantified by plaque assay on Vero cells. Viral burden is reported as log₁₀ PFU/mL. Each data point represents the geometric mean of two independent samples and error bars correspond to one standard deviation. The assay limit of detection is represented by the dashed horizontal line.

Figure 3

Antiviral activity of clinical exposures of interferon-α (IFN) and/or ribavirin (RBV) after 24 h post-infection (p.i.) in the Hollow fiber infection model (HFIM) system. Huh-7 cells were mixed with DENV2 at a multiplicity of infection of 0.001 PFU/cell and inoculated into the HFIM system. After 24 h post-inoculation, cells were exposed to RBV and/or IFN at levels corresponding to the clinical regimen. Supernatants were collected from the system for three days after the start of drug administration. Viral burden was quantified by plaque assay in Vero cells and is described as log₁₀ PFU/mL. Each data point represents the geometric mean of two independent samples and error bars (smaller than symbols and, thus, not visible) correspond to one standard deviation. The dashed horizontal line represents the assay limit of detection.